Objective To study the chemical constituents of Coptosapelta diffusa roots and measure their antimicrobial activities. Methods The chemical constituents were isolated and purified using silica gel, Sephadex LH-20 column chromatographies. Their structures were identified based on spectral analysis, and the antimicrobial activity was carried out by the paper diffusion method. Results 12 compounds were identified as lapachol (1), hydroxyhydrolapachol (2), dehydro-α-lapachone(3), α-lapachone(4), rhinacantin A(5), β-lapachone(6), 2-carbomethoxyanthraquinone(7), 7-hydroxy-2-methylanthraquinone (8), isoscopoletin (9), alyxialactone (10), (3S)-3-(hydroxymethyl)-3H-isobenzofuran-1-one (11), and (7’S, 8S, 8’R)-4, 4’-dihydroxy-3, 3’, 5, 5’-tetramethoxy-7’, 9-epoxylignan-9’-ol-7-one(12). Compounds 1-4 and 6 showed good antimicrobial activity against the tested strains. Conclusion Compounds 4-6 and 10-12 were isolated from this plant for the first time. Lapachol was identified as the main constituent of C. diffusa roots with the content of 3.56 g/kg (dry weight), indicating that C. diffusa roots could serve as a source of lapachol. This study provided a scientific basis for the folk antimicrobial and anti-inflammatory medicinal function of C. diffusa roots.